In recent years, the scientific and medical communities have given increased attention to various polypeptides useful as therapeutic agents and to methods of isolating such polypeptides from the complex source materials in which they are present. An example of such a polypeptide is a blood factor obtained from plasma known as the antihemophilic factor. This blood factor is also identified as Factor VIII procoagulant activity protein (Factor VIII:C). This protein acts to correct the clotting defect in individuals with hemophilia A. It exists in plasma complexed with another protein known as Factor VIII-related protein or Factor VIII:RP. Other designations for Factor VIII:RP are Factor VIII:R:Ag and von Willebrand factor. Because of Factor VIII:C's therapeutic value as a coagulant, it has been regarded as desirable to purify Factor VIII and to isolate Factor VIII:C from Factor VIII:RP. Various procedures have been suggested for the isolation and purification of Factor VIII:C and other polypeptides of therapeutic value. These methods have been generally based on the techniques of immunoaffinity, affinity or ion exchange chromatography. For example, the method in a recent patent for the ultrapurification of Factor VIII:C, Zimmerman & Fulcher, U.S. Pat. No. Re. 32,011, employs such a two-step procedure of affinity and ion-exchange chromatography. Essentially, a Factor-VIII preparation is passed through a column containing agarose beads coupled with mouse monoclonal antibodies directed to Factor VIII:RP. The Factor VIII:C, which is complexed to the von Willebrand factor, is adsorbed onto the matrix while uncomplexed Factor VIII:C moieties and contaminants pass through the column as unbound material. The Factor VIII:C is removed from the bound von Willebrand: antibody complex with a high salt solution containing calcium. The Factor VIII:C solution is desalted and finally adsorbed onto an ion-exchange column, more specifically, agarose beads coupled with positively charged aminohexyl groups. The Factor VIII:C is desorbed from the column with a high salt solution. Although this method is suitable for use in the ultrapurification of a polypeptide, it lacks several important features which would improve the therapeutic safety of the product and facilitate its large-scale production. One distinctive feature of the Zimmerman et al reissue patent is that the monoclonal antibodies are directed to another polypeptide (von Willebrand factor) that is usually in excess and considered associated with the polypeptide of interest (Factor VIII:C). Depending upon the source material, it is possible that as much as 50% of the Factor VIII:C can be in a form not associated with von Willebrand factor. See Amphlett et al, U.S. Pat. No. 4,508,709. The unassociated Factor VIII:C will not be bound by the monoclonal antibodies used in the immunoaffinity step described above, and will consequently be lost in the purification process. To date, there has been no proven advantage for having Factor VIII:C as a product in the uncomplexed form only. Evidence suggests that Factor VIII:C is protected longer from proteolysis by its association with the von Willebrand factor, an important feature when isolating the polypeptide from complex source materials. See Weiss et al, J. Clin., Invest. 60, 390-404, 1977. Hence, instead of being a disadvantage, the association of von Willebrand factor with Factor VIII:C could be beneficial insofar as it may confer stability to Factor VIII:C during the purification steps and may extend the half-life of the polypeptide during its therapeutic administration.
Secondly, monoclonal antibodies covalently coupled to any matrix have a tendency to leach, or separate from their matrix and contaminate the final polypeptide-containing product. The patented procedure described above and in the prior art does not guard against the probability of nonhuman cell-derived leached monoclonal antibodies from the immunoaffinity step accompanying and reassociating with the Factor VIII:C during the second ion-exchange step. The high ionic strength buffer used in the immunoaffinity procedure to elute Factor VIII:C is reduced to low ionic strength, which could allow monoclonal antibodies removed from the immunoaffinity column either to rebind to Factor VIII:C or to bind and desorb from the ion-exchange matrix along with the Factor VIII:C.
Thirdly, the desalting process required for the polypeptide-containing solution prior to loading onto the ion exchange column is usually accomplished by large volume dilution, dialysis, or ultra-filtration molecular washing. These methods are not only cumbersome for large-scale production volumes, but inevitably lead to loss of product.
Finally, the aqueous source materials in which the polypeptides of interest are found often are contaminated with one or more viruses. There are techniques for inactivating viruses in polypeptide mixtures, but attempts to combine such techniques with known polypeptide purification processes have produced methods with a multiplicity of steps unsuitable for large-volume production. The methods have also frequently been only partially successful in purifying the polypeptide. For example, in the prior art, a number of viral-inactivating agents have been shown effective for inactivating viruses. The agents have, however, been either denaturing or difficult to separate from the polypeptide of interest, and have required a special treatment or separation step. Other conventional methods for treating polypeptide-containing preparations for potential viral contamination, such as heat or irradiation, have resulted in either significant denaturation of the polypeptide of interest and/or insufficient inactivation of viruses.
The use of viral inactivating agents described herein can inactivate viruses without adversely affecting the biological activity of the polypeptide of interest. Treatment of the aqueous source materials with the viral inactivating agents, accompanied by the other procedures of this invention, produces a final product substantially free of viruses as well as viral inactivating agents.
It is a principal object of this invention to provide a purification process for polypeptides particularly adapted to large-scale purification of a polypeptide with a low level of polypeptide denaturation. It is another object of the invention to provide a polypeptide purification process which is also effective in reducing antibody and viral contamination of the purified product. One additional object of this invention is the development of a process for purifying a polypeptide, free of contaminating substances such as other proteins, viruses, and treating agents used in the purification steps.